A Silicon-Stereogenic Silanol - 18 O-Isotope Labeling and Stereogenic Probe Reveals Hidden Stereospecific Water Exchange Reaction.

A silicon-stereogenic aminosilanol was isolated in excellent diastereomeric ratio and the absolute configuration was determined. The silanol is configurative and condensation stable in solution and shows stereoselective transformations with a clean stereospecific pathway in follow-up reactions, which leads to the isolation of a silicon-stereogenic zinc complex and siloxane compounds. Investigations with 18 O-labelled water and mass spectrometry analysis revealed an otherwise hidden exchange of oxygen atoms of silanol and water in solution that proceeds with retention of the configuration at the silicon center. This novel combination of a stereochemical probe and isotopic labeling in a silicon-stereogenic compound opens new analytic possibilities to study stereochemical courses of reactions with the aid of chiral silanols mechanistically.

Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity. METHODS: We performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes. RESULTS: Prior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity. CONCLUSIONS: APAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication. LAY SUMMARY: Only one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine.

Production of Siderophores by an Apple Root-Associated Streptomyces ciscaucasicus Strain GS2 Using Chemical and Biological OSMAC Approaches.

Apple Replant Disease (ARD) is a significant problem in apple orchards that causes root tissue damage, stunted plant growth, and decline in fruit quality, size, and overall yield. Dysbiosis of apple root-associated microbiome and selective richness of Streptomyces species in the rhizosphere typically concurs root impairment associated with ARD. However, possible roles of Streptomyces secondary metabolites within these observations remain unstudied. Therefore, we employed the One Strain Many Compounds (OSMAC) approach coupled to high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSn) to evaluate the chemical ecology of an apple root-associated Streptomycesciscaucasicus strain GS2, temporally over 14 days. The chemical OSMAC approach comprised cultivation media alterations using six different media compositions, which led to the biosynthesis of the iron-chelated siderophores, ferrioxamines. The biological OSMAC approach was concomitantly applied by dual-culture cultivation for microorganismal interactions with an endophytic Streptomyces pulveraceus strain ES16 and the pathogen Cylindrocarpon olidum. This led to the modulation of ferrioxamines produced and further triggered biosynthesis of the unchelated siderophores, desferrioxamines. The structures of the compounds were elucidated using HRMSn and by comparison with the literature. We evaluated the dynamics of siderophore production under the combined influence of chemical and biological OSMAC triggers, temporally over 3, 7, and 14 days, to discern the strain's siderophore-mediated chemical ecology. We discuss our results based on the plausible chemical implications of S. ciscaucasicus strain GS2 in the rhizosphere.

Anti-inflammatory Flavanones and Flavones from Tephrosia linearis.

Phytochemical analysis of a methanol-dichloromethane (1:1) extract of the aerial parts of Tephrosialinearis led to the isolation of 18 compounds. Seven of these, namely, lineaflavones A-D (1-4), 6-methoxygeraldone (5), 8″-acetylobovatin (6), and 5-hydroxy-7-methoxysaniculamin A (7) are new compounds. The compounds were characterized based on their NMR and HRMSn data. The anti-inflammatory effects of the crude extract and isolated compounds were evaluated by measuring the levels of interleukins (IL-1ß, IL-2, and IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) in lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs). The crude extract inhibited the release of all cytokines except IL-1ß, which slightly increased in comparison to the LPS control. All the tested compounds suppressed the production of IL-2, GM-CSF, and TNF-α. Whereas compounds 1, 2, 4-8, 10-15, 17, and 18 decreased production of IL-6, compounds 1, 2, 4, 7, 10, 13-15, and 17 inhibited the release of IL-1ß. It is worth noting that most of the compounds tested showed a superior reduction in cytokines release compared to the reference drug ibuprofen.

Apple Replant Disease: Causes and Mitigation Strategies.

After replanting apple (Malus domestica Borkh.) on the same site severe growth suppressions, and a decline in yield and fruit quality are observed in all apple producing areas worldwide. The causes of this complex phenomenon, called apple replant disease (ARD), are only poorly understood up to now which is in part due to inconsistencies in terms and methodologies. Therefore we suggest the following definition for ARD: ARD describes a harmfully disturbed physiological and morphological reaction of apple plants to soils that faced alterations in their (micro-) biome due to the previous apple cultures. The underlying interactions likely have multiple causes that extend beyond common analytical tools in microbial ecology. They are influenced by soil properties, faunal vectors, and trophic cascades, with genotype-specific effects on plant secondary metabolism, particularly phytoalexin biosynthesis. Yet, emerging tools allow to unravel the soil and rhizosphere (micro-) biome, to characterize alterations of habitat quality, and to decipher the plant reactions. Thereby, deep insights into the reactions taking place at the root rhizosphere interface will be gained. Counteractions are suggested, taking into account that culture management should emphasize on improving soil microbial and faunal diversity as well as habitat quality rather than focus on soil disinfection.

Spatio-temporal visualization of the distribution of acetaminophen as well as its metabolites and adducts in mouse livers by MALDI MSI.

Acetaminophen (APAP) is one of the most intensively studied compounds that causes hepatotoxicity in the pericentral region of the liver lobules. However, spatio-temporal information on the distribution of APAP, its metabolites and GSH adducts in the liver tissue is not yet available. Here, we addressed the question, whether APAP-GSH adducts and GSH depletion show a zonated pattern and whether the distribution of APAP and its glucuronide as well as sulfate conjugates in liver lobules are zonated. For this purpose, a matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) technique was established, where the MSI images were superimposed onto CYP2E1 immunostained tissue. A time-dependent analysis (5, 15, 30, 60, 120, 240, 480 min) after intraperitoneal administration of 300 mg/kg APAP and a dose-dependent analysis (56 up to 500 mg APAP/kg) at 30 min were performed. The results demonstrate that the MALDI MSI technique allows the assignment of compounds and their metabolites to specific lobular zones. APAP-GSH adducts and GSH depletion occurred predominantly in the CYP2E1-positive zone of the liver, although GSH also decreased in the periportal region. In contrast, the parent compound, APAP sulfate and APAP glucuronide did not show a zonated pattern and tissue concentrations showed a similar time course as the corresponding analyses were performed with blood from the portal and liver veins. In conclusion, the present study is in agreement with the concept that pericentral CYPs form NAPQI that in the same cell binds to and depletes GSH but a lower level of GSH adducts is also observed in the periportal region. The results also provide further evidence of the recently published concept of 'aggravated loss of clearance capacity' according to which also liver tissue that survives intoxication may transiently show decreased metabolic capacity.

Synthetic Origin of Tramadol in the Environment.

The presence of tramadol in roots of Sarcocephalus latifolius trees in Northern Cameroon was recently attributed to point contamination with the synthetic compound. The synthetic origin of tramadol in the environment has now been unambiguously confirmed. Tramadol samples isolated from tramadol pills bought at a street market in downtown Maroua and highly contaminated soil at Houdouvou were analyzed by high-precision (14)C measurements by accelerator mass spectrometry ((14)C AMS): Tramadol from the pills did not contain any radiocarbon, thus indicating that it had been synthesized from (14)C-free petroleum-derived precursors. Crucially, tramadol isolated from the soil was also radiocarbon-free. As all biosynthetic plant compounds must contain radiocarbon levels close to that of the contemporary environment, these results thus confirm that tramadol isolated from the soil cannot be plant-derived. Analyses of S. latifolius seeds, in vitro grown plants, plants from different origins, and stable-isotope labeling experiments further confirmed that synthetic tramadol contaminates the environment.

Antiplasmodial and Cytotoxic Triterpenoids from the Bark of the Cameroonian Medicinal Plant Entandrophragma congoënse.

Eight new triterpenoids, prototiamins A-G (1-6, 9) and seco-tiaminic acid A (10), were isolated along with four known compounds from the bark of Entandrophragma congoënse. Their structures were elucidated by means of HRMS and different NMR techniques and chemical transformations. Assignments of relative and absolute configurations for the new compounds were achieved using NOESY experiments and by chemical modification including the advanced Mosher's method. Additionally, the structure and relative configuration of compound 3 were confirmed by single-crystal X-ray diffraction analysis. Compounds 1, 3, and 5 displayed significant in vitro antiplasmodial activity against the erythrocytic stages of chloroquine-sensitive Plasmodium falciparum strain NF54. Prototiamin C (3) was the most potent of the compounds isolated, with an IC50 value of 0.44 µM. All compounds tested showed low cytotoxicity for the L6 rat skeletal myoblast cell line.

Shifts in abundance and diversity of mobile genetic elements after the introduction of diverse pesticides into an on-farm biopurification system over the course of a year.

Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1ß and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1ß plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

Endophytes are hidden producers of maytansine in Putterlickia roots.

Several recent studies have lent evidence to the fact that certain so-called plant metabolites are actually biosynthesized by associated microorganisms. In this work, we show that the original source organism(s) responsible for the biosynthesis of the important anticancer and cytotoxic compound maytansine is the endophytic bacterial community harbored specifically within the roots of Putterlickia verrucosa and P. retrospinosa plants. Evaluation of the root endophytic community by chemical characterization of their fermentation products using HPLC-HRMS(n), along with a selective microbiological assay using the maytansine-sensitive type strain Hamigera avellanea revealed the endophytic production of maytansine. This was further confirmed by the presence of AHBA synthase genes in the root endophytic communities. Finally, MALDI-imaging-HRMS was used to demonstrate that maytansine produced by the endophytes is typically accumulated mainly in the root cortex of both plants. Our study, thus, reveals that maytansine is actually a biosynthetic product of root-associated endophytic microorganisms. The knowledge gained from this study provides fundamental insights on the biosynthesis of so-called plant metabolites by endophytes residing in distinct ecological niches.

Determination of anxiolytic veterinary drugs from biological fertilizer blood meal using liquid chromatography high-resolution mass spectrometry.

A liquid environment-friendly agricultural material originating from animal blood, blood meal, was employed to detect anxiolytic veterinary drugs using a combination of liquid-liquid extraction (LLE) and positive electrospray ionization Orbitrap mass spectrometry. Every positive ion of the analytes was consistent with [M+H](+) , and the accurate mass analysis and mass spectral filtration with a 2-ppm mass tolerance window were applied to identify and quantitate the analytes and metabolites. The developed LLE method was validated with the lowest calibrated level, linearity (r(2) ), recovery, repeatability and the within-laboratory reproducibility, which were in the ranges of 0.3-1 µg/L, 0.9963-0.9995, 48.3-117.5%, 1.1-12.6% and 2.3-15.7%, respectively. The LLE method was compared with a solid-phase extraction (SPE) method; however, its recoveries were <70% for most of the analytes despite good repeatability of 1.2-7.4%. The analytes and the ascertained acepromazine, azaperone and xylazine metabolites were monitored in four actual liquid blood meal samples, and none of the targeted compounds were observed.

Tramadol--a true natural product?

We have independently investigated the source of tramadol, a synthetic analgesic largely used for treating moderate to severe pain in humans, recently found in the roots of the Cameroonian medicinal plant, Nauclea latifolia. We found tramadol and its three major mammalian metabolites (O-desmethyltramadol, N-desmethyltramadol, and 4-hydroxycyclohexyltramadol) in the roots of N. latifolia and five other plant species, and also in soil and local water bodies only in the Far North region of Cameroon. The off-label administration of tramadol to cattle in this region leads to cross-contamination of the soil and water through feces and urine containing parent tramadol as well as tramadol metabolites produced in the animals. These compounds can then be absorbed by the plant roots and also leached into the local water supplies. The presence of tramadol in roots is, thus, due to an anthropogenic contamination with the synthetic compound.

Enantioselective degradation of the chiral fungicides metalaxyl and furalaxyl by Brevibacillus brevis.

For almost four decades, the chiral fungicides metalaxyl and furalaxyl have been in use in plant protection on a global scale. Both substances are distributed as racemic mixtures, yet the desirable interference in nucleic acid synthesis of harmful fungi only occurs by the (-)-R-enantiomer. As enantioselective degradation in Scheyern (Germany) and Yaoundé (Cameroon) soils has been documented, the influence of 50 isolated microorganisms on the R/S ratio was investigated. A high-pressure liquid chromatography method with a chiral column to separate enantiomers of metalaxyl and furalaxyl, and subsequent detection by tandem mass spectrometry, was employed. Only one of these microorganisms, a strain of Brevibacillus brevis, showed an enantioselective degradation pattern in liquid culture; the respective (-)-R-enantiomers were preferably degraded. Moreover, (-)-R-furalaxyl was degraded faster in cultures supplemented simultaneously with both fungicides of the same concentration.

Evaluation of sorption-desorption processes for metalaxyl in natural and artificial soils.

The main process controlling soil-pesticide interaction is the sorption-desorption as influenced by active soil surfaces. The sorption phenomena can influence translocation, volatility, persistence and bioactivity of a pesticide in soil. The present investigation was conducted on natural and artificial soils in order to enumerate the effect of soil components such as montmorillonite and ferrihydrite on the sorption behaviour of the fungicide metalaxyl and if sorption-desorption of the chiral pesticide affects the enantiomeric ratio. The sorption-desorption characteristics of metalaxyl were investigated by batch equilibration technique in a natural soil, two artificial soils, and in pure montmorillonite and ferrihydrite. After extraction, pesticide residues were analyzed by conventional and chiral chromatography using tandem mass spectrometry. A KdSorp (2.3-6.5) suggests low level sorption of metalaxyl with an appreciable risk of run-off and leaching. Thus, metalaxyl poses a threat to surface and ground water contamination. Furthermore, desorption tests revealed a hysteretic effect (H ≤ 0.8) in natural and artificial soils. Significant amount of metalaxyl was found tightly bound to the adsorbents without desorbing readily after desorption cycle. Desorption of 22-56% of the total amount of the retained metalaxyl was determined. This study reveals that an artificial soil derived from different soil constituents can be used to assess their influence on sorption/desorption processes. The present investigation showed that both montmorillonite and ferrihydrite play a significant role in the sorption of metalaxyl. The sorption doesn't influence the enantiomeric ratio of racemic metalaxyl.

In vitro to in vivo acetaminophen hepatotoxicity extrapolation using classical schemes, pharmacodynamic models and a multiscale spatial-temporal liver twin.

In vitro to in vivo extrapolation represents a critical challenge in toxicology. In this paper we explore extrapolation strategies for acetaminophen (APAP) based on mechanistic models, comparing classical (CL) homogeneous compartment pharmacodynamic (PD) models and a spatial-temporal (ST), multiscale digital twin model resolving liver microarchitecture at cellular resolution. The models integrate consensus detoxification reactions in each individual hepatocyte. We study the consequences of the two model types on the extrapolation and show in which cases these models perform better than the classical extrapolation strategy that is based either on the maximal drug concentration (Cmax) or the area under the pharmacokinetic curve (AUC) of the drug blood concentration. We find that an CL-model based on a well-mixed blood compartment is sufficient to correctly predict the in vivo toxicity from in vitro data. However, the ST-model that integrates more experimental information requires a change of at least one parameter to obtain the same prediction, indicating that spatial compartmentalization may indeed be an important factor.

Draft Genome Sequence of the Bacterial Endophyte Priestia megaterium B1, Isolated from Roots of Apple (Malus domestica).

Over the past years, a number of important traits supporting plant growth have been shown for different strains of Priestia megaterium (formerly known as Bacillus megaterium). Here, we report the draft genome sequence of the endophytic bacterial strain Priestia megaterium B1, which was isolated from surface-sterilized roots of apple plants.

Cytotoxic and genotoxic properties of artathomsonine, a new oxoberberine alkaloid from Artabotrys thomsonii (annonaceae).

A phytochemical investigation of the liana of Artabotrys thomsonii led to the isolation of a new oxoberberine alkaloid, 2,10-dihydroxy-3,9-dimethoxy-8-oxo-protoberberine (7), along with six known compounds. Their chemical structures were elucidated by 1 D and 2 D NMR spectroscopic methods and HRESI-MSn data analysis. Compounds 4 and 7 were selected for further in vitro investigations. In accordance with expectations from their chemical structures, compounds 7 and 4 showed a clear antioxidant activity in a cell-free assay, with compound 7 being 7-fold more active than 4. Cytotoxicity, cytostatic and genotoxic effects only occurred at high micromolar concentrations of 50 µM or more. Compound 7 was slightly less effective than compound 4. A low micromolar concentration of 10 µM did not cause any damaging cellular effects but showed potential for a protection against the micronucleus-inducing effect of reactive oxygen species hydrogen peroxide, although not to a significant extent.

Effect of artificial reconstitution of the interaction between the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin biosynthesis.

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied and occasionally produce host-specific compounds. We recently isolated an endophytic fungus, Fusarium solani, from Camptotheca acuminata, capable of biosynthesizing camptothecin (CPT, 1), but this capability substantially decreased on repeated subculturing. The endophyte with an impaired 1 biosynthetic capability was artificially inoculated into the living host plants and then recovered after colonization. Although the host-endophyte interaction could be reconstituted, biosynthesis of 1 could not be restored. Using a homology-based approach and high-precision isotope-ratio mass spectrometry (HP-IRMS), a cross-species biosynthetic pathway is proposed where the endophyte utilizes indigenous G10H (geraniol 10-hydroxylase), SLS (secologanin synthase), and TDC (tryptophan decarboxylase) to biosynthesize precursors of 1. However, the endophyte requires host STR (strictosidine synthase) in order to condense the nitrogen-containing moiety (tryptamine, 2) with the carbon-containing moiety (secologanin, 3) to form strictosidine (4) and complete the biosynthesis of 1. Biosynthetic genes of 1 in the seventh subculture generation of the endophyte revealed random and unpredictable nonsynonymous mutations. These random base substitutions led to dysfunction at the amino acid level. The controls, Top1 gene and rDNA, remained intact over subculturing, revealing that instability of biosynthetic genes of 1 was not reflected in the primary metabolic processes and functioning of the housekeeping genes. The present results reveal the causes of decreased production of 1 on subculturing, which could not be reversed by host-endophyte reassociation.

Chemometric evaluation of the anti-cancer pro-drug podophyllotoxin and potential therapeutic analogues in Juniperus and Podophyllum species.

INTRODUCTION: Podophyllotoxin, deoxypodophyllotoxin, demethylpodophyllotoxin and podophyllotoxone are four therapeutically potent secondary metabolites. There is a dearth of information on the holistic analysis of their distribution pattern in both phylogenetic and ecological contexts. OBJECTIVES: To analyse the continuum of the above metabolites in Juniperus and Podophyllum species collected from natural populations in Himalayan environments and the botanical gardens of Rombergpark and Haltern (Germany) using multi-component LC-ESI-MS/MS, coupled with statistically relevant chemometric assessment. METHODOLOGY: We evaluated the individual and holistic metabolite profiles and chemometrically correlated the phytochemical loads between various species (infraspecific), organic and aqueous extracts, and populations of the same species from different locations, different species from same location, different species from different locations and infrageneric populations from same and different locations. RESULTS: Multivariate analysis revealed Juniperus x-media Pfitzeriana as a suitable alternative to Podophyllum hexandrum for commercial exploitation. A significant positive correlation of podophyllotoxone with both podophyllotoxin and demethylpodophyllotoxin, and a negative correlation of podophyllotoxin with both deoxypodophyllotoxin and demethylpodophyllotoxin (infraspecific among Podophyllum), were observed by Kruskal's multidimensional scaling and corroborated by principal component analysis, indicating probable similarity and/or difference between the biosynthetic pathways, and synergistic and/or antagonistic principles, respectively. Finally, linear discriminant analysis and hierarchical agglomerative cluster analysis revealed considerable infrageneric and infraspecific variability in secondary compound spectra and load of the different populations under study. CONCLUSION: Such holistic studies of plants and their therapeutic metabolites ought to assist in selecting plants, geographical areas and environmental conditions for bioprospecting and global-scale phytochemical and phylogenetic diversity studies in the future.

Impact of Nonylphenols and Polyhalogenated Compounds in Follicular Fluid on the Outcome of Intracytoplasmic Sperm Injection.

Endocrine-disrupting chemicals (EDCs) interfere with the mammalian hormone system and alter its endo- and paracrine regulation. The goal of the present study was to examine the presence of 14 EDCs, including the technical mixture of nonylphenols and Mirex, in human follicular fluid (FF) and to find a potential correlation between endocrine active substances and a possible impact on female fertility. Furthermore, potential sources of EDC exposition regarding patients' lifestyle and socioeconomic factors were investigated. Human FF was collected from a total of 210 women undergoing intracytoplasmic sperm injection-treatment cycles because of male subfertility. The presence of EDCs was analyzed using gas chromatography coupled with mass spectrometry. Thirteen of the 14 investigated EDCs were present in every FF sample; compounds with the highest concentrations in FF were nonylphenol and Mirex. Nearly all kinds of EDCs led to significantly reduced maturation and fertilization rate. No significant influence of EDC concentration on the clinical pregnancy rate was observed for neither of the analyzed EDCs. Patients who obtained their clothes and textiles at fashion discounters displayed a higher amount of EDCs in their FF. In contrast, patients' residential area, source of food products, and nicotine or caffeine consumed were not associated with EDC accumulation. Clinicaltrials.gov NCT01385605 (11 July 2011).